Oligodendrocyte precursor cell and method for culturing oligodendrocytes

Abstract

The present invention relates to a method for culturing oligodendrocytes, the method comprising: a step for adding an isolation medium to a suspension containing neural stem cells to dissociate tissue; a step for resuspending the dissociated cell suspension by adding a first solution; a step for adding a first solution to the resuspended solution and performing primary centrifugation to obtain a supernatant; a step for adding a second solution to the supernatant and performing secondary centrifugation to obtain a cell pellet; a step for resuspending the cell pellet and then plating same; and a step for culturing the plated cells in a proliferation medium.

Description

Method for culturing oligodendrocyte precursor cells and oligodendrocytes

[0001] The present invention relates to a method for culturing oligodendrocyte precursor cells and oligodendrocytes.

[0002] Oligodendrocytes are cells that form myelin in the central nervous system and play a very important role in saccadic conduction. Various studies are being conducted on myelin formation during development and its role in brain diseases.

[0003] Oligodendrocytes are known to undergo several stages of maturation from precursor cells (oligodendrocyte precursor cells or oligodendrocyte progenitor cells) to become myelin-forming mature oligodendrocytes, and these various stages coexist in vivo. Therefore, it is difficult to determine the degree of maturation from oligodendrocyte precursor cells to myelin-forming mature oligodendrocytes at each stage through in vivo experiments alone. Since each maturation stage has different vulnerabilities to damage such as ischemia or neuroinflammation, cell experiments are preferred depending on the purpose of the experiment.

[0004] Immortalized oligodendrocyte cell lines may differ from primary cell lines in their maturation stages, and because they are immortalized cells, their vulnerability to damage is difficult to assess. Therefore, primary cultures, obtained directly from the brain, are preferred for most studies on oligodendrocyte progenitor cells and oligodendrocytes.

[0005] Currently, methods for obtaining primary cultured oligodendrocyte progenitor cells can be broadly categorized into two: antibody-based methods and physical-based methods. Antibody-based methods include immunopanning, which involves obtaining cell-selective antibodies from hybridomas and coating cell culture dishes to obtain oligodendrocyte progenitor cells. Furthermore, the MACS method utilizes antibodies bound to magnetic beads. However, these methods require multiple steps, expensive equipment, and expensive magnetic bead-bound antibodies.

[0006] Methods that utilize the physical properties of cells include the shaking method, which has been used for the longest time, using cell adhesion, and the density gradient method, which uses cell specific gravity. Although the shaking method has been used for the longest time, it has the disadvantage of requiring an experienced experimenter and taking a long time to obtain cells. In the case of the density gradient method, there are various methods, from separation using Percoll, which has concerns about cytotoxicity, to the recent Iodixanol (OptiPrep TM ) have been proposed, but there are disadvantages such as creating multiple layers to implement multiple densities, the influx of other cells when selectively acquiring one layer among multiple layers, and the fact that the amount of tissue that can be separated by centrifugation is greatly limited due to the characteristic of cells being concentrated on one plane during the density gradient centrifugation process.

[0007] Therefore, there is a need to develop a method for culturing oligodendrocyte precursor cells and oligodendrocytes that minimizes the influx of other cells economically in a relatively short period of time.

[0008] The background technology of this application, Republic of Korea Patent Publication No. 10-2020-0136452, relates to a method for producing differentiation medium and oligodendrocyte precursors.

[0009] The present invention is intended to solve the problems of the above-mentioned prior art and provides a method for culturing oligodendroglial precursor cells.

[0010] Additionally, a method for culturing oligodendrocytes is provided.

[0011] In addition, a method for culturing myelin-forming oligodendrocytes is provided.

[0012] However, the technical tasks to be achieved by the embodiments of the present invention are not limited to the technical tasks described above, and other technical tasks may exist.

[0013] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a method for culturing oligodendrocyte precursor cells, comprising the steps of: dissociating tissue by adding isolation media to a suspension containing neural stem cells; resuspending the dissociated cell suspension by adding a first solution; further adding the first solution to the resuspended solution and performing a first centrifugation to obtain a supernatant; adding a second solution to the supernatant and performing a second centrifugation to obtain a cell pellet; resuspending the cell pellet and then plating it; and culturing the plated cells in a proliferation media.

[0014] According to one embodiment of the present invention, the separation medium may include, but is not limited to, Hibernate-A medium, GlutaMAX, Penicillin / Streptomycin, and papain.

[0015] According to one embodiment of the present invention, the first solution may include, but is not limited to, a separation medium and high-density iodixanol.

[0016] According to one embodiment of the present invention, the second solution may include, but is not limited to, a separation medium and low-density iodixanol.

[0017] According to one embodiment of the present invention, the proliferation medium may include, but is not limited to, DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) medium containing growth factors PDGF-AA, FGF, and EGF.

[0018] According to one embodiment of the present invention, the growth medium may further include a supplement selected from the group consisting of, but not limited to, B27, GlutaMAX, Penicillin / Streptomycin, and combinations thereof.

[0019] In addition, the second aspect of the present invention provides a method for culturing oligodendrocytes, comprising: a step of culturing oligodendrocyte precursor cells by the method according to the first aspect of the present invention; a step of isolating and replating the oligodendrocyte precursor cells by treating them with an enzyme; and a step of culturing the plated oligodendrocyte precursor cells in a proliferation medium.

[0020] According to one embodiment of the present invention, the enzyme may include, but is not limited to, one selected from the group consisting of Accutase, TrypLE, trypsin, and combinations thereof.

[0021] According to one embodiment of the present invention, the proliferation medium may include, but is not limited to, DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) medium containing growth factors PDGF-AA and FGF.

[0022] According to one embodiment of the present invention, the growth medium may further include a supplement selected from the group consisting of, but not limited to, B27, GlutaMAX, Penicillin / Streptomycin, and combinations thereof.

[0023] In addition, the third aspect of the present invention provides a method for culturing myelin-forming oligodendrocytes, comprising the steps of culturing oligodendrocytes by the method according to the second aspect of the present invention; and the step of myelinating the oligodendrocytes by replacing the medium with a differentiation medium.

[0024] According to one embodiment of the present invention, the differentiation medium may include, but is not limited to, a neurobasal medium containing growth factors N2 and T3.

[0025] According to one embodiment of the present invention, the differentiation medium may further include a supplement selected from the group consisting of, but not limited to, B27, GlutaMAX, Penicillin / Streptomycin, and combinations thereof.

[0026] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0027] The culture method of oligodendrocyte precursor cells and oligodendrocytes according to the present invention borrows from the conventional density gradient-based method, but instead of performing layering, it adopts a method of two sequential centrifugations to increase the efficiency of tissue use, and by eliminating the effort and experimenter skill required to form multiple layers, even inexperienced experimenters can obtain myelin-forming oligodendrocytes in a relatively short period of time. This not only increases the ease of various experiments using primary cultured oligodendrocyte precursor cells and oligodendrocytes, but also provides the possibility of obtaining adult-derived oligodendrocyte precursor cells in the future, which can be of great help in studying functional changes in oligodendrocyte precursor cells and oligodendrocytes in various degenerative diseases.

[0028] In addition, the method according to the present invention can minimize irregular cell arrangement caused by the proliferation and migration ability of oligodendrocyte precursor cells and narrow intercellular gaps that cause cell death during differentiation by performing replating.

[0029] In addition, the culture method according to the present invention promotes the proliferation of oligodendrocyte precursor cells by optimizing the medium conditions, thereby significantly increasing the number of cells obtained, and compared to the DMEM-based medium conditions that were widely used in the past for differentiation into myelin-forming oligodendrocytes, more morphologically mature oligodendrocytes can be obtained during the same differentiation period.

[0030] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0031] Figure 1a is a schematic diagram of a culture method according to an experimental example of the present invention, Figure 1b is a representative image of cells isolated after 5 days of proliferation (immunostaining for NG2 and A2B5, P: PDGF, F: FGF, E: EGF), Figure 1c is a representative image of cells isolated after 5 days of proliferation (immunostaining for Tuj1, P: PDGF, F: FGF, E: EGF), Figure 1d is the result of quantification of the cell ratio after 5 days of proliferation according to the medium composition corresponding to the medium conditions of Table 1 [10 images per media condition (6 media conditions, n=3 samples), approximately 300 DAPI+ cells per image. One-way ANOVA and Tukey's multiple comparison test, ***: P < .001, ns: not statistically significant (P > .05)].

[0032] Figure 2a is a representative image of cells isolated after 5 days of proliferation according to one experimental example of the present invention (staining for EdU, Ki67. P: PDGF, F: FGF, E: EGF), Figure 2b is the quantification result of EdU+ / DAPI+ (%) cells according to the media composition corresponding to the media conditions of Table 1 [10 images per media condition (6 media conditions, n=3 samples), approximately 500 DAPI+ cells per image. One-way ANOVA and Tukey's multiple comparison test, ns: not statistically significant (P > .05)], Figure 2c is the quantification result of Ki67+ / DAPI+ (%) cells according to the media composition corresponding to the media conditions of Table 1 [10 images per media condition (6 media conditions, n=3 samples), approximately 500 DAPI+ cells per image. One-way ANOVA and Tukey's multiple comparison test, ns: not statistically significant (P > .05)], Figure 2d is a graph of the results of the WST-8 viability assay, values ​​for the DMEM (medium composition 1) group cultured for 72 hours (6 medium conditions, n=3 samples, one-way ANOVA and Tukey's multiple comparison test, ***: P < .001).

[0033] Figure 3a is a representative image of cells isolated 4 days after differentiation according to one experimental example of the present invention (immunostaining for myelin basic protein (MBP)), Figure 3b is a representative image of cells isolated at the periphery of colonies 4 days after differentiation (region B of Figure 3a; immunostaining for cleaved caspase 3, MBP), Figure 3c is a representative image of cells isolated at the center of colonies 4 days after differentiation (region C of Figure 3a; immunostaining for cleaved caspase 3, MBP), Figure 3d is a scatter plot of the average nearest neighbor distance of cells in the immunostaining images and the percentage of cleaved caspase-3+ / DAPI+ cells and the percentage of cell death measured by spline / LOWESS fitting. The correlation between average nearest neighbor distance and cell death rate was analyzed by simple linear regression for each media condition [10 images per media condition (6 media conditions, n=3 samples), approximately 300 DAPI+ cells per image].

[0034] Figure 4a is a schematic diagram of a revised culture procedure including passage of expanded OPCs according to one experimental example of the present invention, Figure 4b is a representative image of passaged and redistributed OPCs (immunostaining for Olig2; arrows indicate Olig2+ cells with atypical morphology), Figure 4c is the quantification of Olig2+ / DAPI+ (%) cells according to the media composition corresponding to the media conditions in Table 1 [(10 images per media condition (6 media conditions, n=3 samples), approximately 50 DAPI+ cells per image, one-way ANOVA and Tukey's multiple comparison test, ns: not statistically significant (P > .05)), Figure 4d is a representative histogram of nearest neighbor distance (NND) of OPCs after passage [10 images per media condition (6 media conditions, n=3 samples), approximately 200 DAPI+ cells per image], Figure 4e is a spatial distribution of passaged OPCs according to proliferation media composition. Here, G-function (cumulative distribution of nearest neighbor distances) graph (gray area indicates the boundary of complete spatial randomness), Fig. 4f is a representative low-magnification image of passaged OPCs 4 days after differentiation into mature oligodendrocytes in neurobasal-based differentiation medium (condition 5 in Table 2; immunostaining for cleaved caspase 3, MBP), and Fig. 4g is a scatterplot of the mean nearest neighbor distance of cells in the immunostained images versus the percentage of cleaved caspase 3+ / DAPI+ cells and the percentage of apoptosis measured by spline / LOWESS fitting. The correlation between mean nearest neighbor distance and percentage of apoptosis was analyzed by simple linear regression for each media condition [9 images per media condition (6 media conditions, n=3 samples), approximately 200 DAPI+ cells per image].

[0035] Figure 5a is a representative image of differentiated oligodendrocytes after passage for 2 or 4 days in the designated differentiation medium composition according to one experimental example of the present invention (immunostaining for MBP), Figure 5b is a bar graph of the cell composition of differentiated oligodendrocytes according to the medium composition corresponding to the medium conditions of Table 2, and Figure 5c is the result of Sholl analysis of differentiated oligodendrocytes [Comparison of morphological complexity was performed by comparing the total number of intersections per cell; 10 images per media condition (6 media conditions, n=3 samples); approximately 100 MBP+ OL per image; one-way ANOVA and Tukey's multiple comparison test; ****: P < .0001; ns: not statistically significant (P > .05)].

[0036] Figure 6a is a representative image of a neuron / oligodendrocyte co-culture according to an experimental example of the present invention (immunostaining for MBP, Tuj1), and Figure 6b is a representative image of differentiated oligodendrocytes cultured on aligned nanofibers (immunostaining for MBP).

[0037] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0038] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0039] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0040] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0041] The terms "about," "substantially," and the like, as used herein, are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings referred to are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that contain precise or absolute numerical values ​​to aid understanding of the present disclosure. Furthermore, throughout the present disclosure, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0042] Throughout this specification, the term "combination thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0043] Throughout this specification, references to “A and / or B” mean “A, B, or A and B.”

[0044] Hereinafter, the culture method of oligodendrocyte precursor cells, oligodendrocytes, and myelin-forming oligodendrocytes of the present invention will be described in detail with reference to embodiments, examples, and drawings. However, the present invention is not limited to these embodiments, examples, and drawings.

[0045]

[0046] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a method for culturing oligodendrocyte precursor cells, comprising the steps of: dissociating tissue by adding isolation media to a suspension containing neural stem cells; resuspending the dissociated cell suspension by adding a first solution; further adding the first solution to the resuspended solution and performing a first centrifugation to obtain a supernatant; adding a second solution to the supernatant and performing a second centrifugation to obtain a cell pellet; resuspending the cell pellet and then plating it; and culturing the plated cells in a proliferation media.

[0047] The culture method of oligodendrocyte precursor cells and oligodendrocytes according to the present invention borrows from the conventional density gradient-based method, but instead of performing layering, it adopts a method of two sequential centrifugations to increase the efficiency of tissue use, and by eliminating the effort and experimenter skill required to form multiple layers, even inexperienced experimenters can obtain myelin-forming oligodendrocytes in a relatively short period of time. This not only increases the ease of various experiments using primary cultured oligodendrocyte precursor cells and oligodendrocytes, but also provides the possibility of obtaining adult-derived oligodendrocyte precursor cells in the future, which can be of great help in studying functional changes in oligodendrocyte precursor cells and oligodendrocytes in various degenerative diseases.

[0048] To culture oligodendrocyte precursor cells, the tissue is first dissociated by adding isolation media to the suspension containing neural stem cells.

[0049] According to one embodiment of the present invention, the separation medium may include, but is not limited to, Hibernate-A medium, GlutaMAX, Penicillin / Streptomycin, and papain.

[0050] Next, the first solution is added to the dissociated cell suspension to resuspend it.

[0051] According to one embodiment of the present invention, the first solution may include, but is not limited to, a separation medium and high-density iodixanol.

[0052] The above high-density iodixanol has a higher density than the low-density iodixanol described below, and preferably, the high-density iodixanol may be 12% Optiprep, but is not limited thereto.

[0053] Next, the first solution is added to the resuspended solution and centrifuged for the first time to obtain the supernatant.

[0054] Next, a second solution is added to the supernatant and centrifuged a second time to obtain a cell pellet.

[0055] According to one embodiment of the present invention, the second solution may include, but is not limited to, a separation medium and low-density iodixanol.

[0056] The above low-density iodixanol has a lower density than the above high-density iodixanol, and preferably, the low-density iodixanol may be 6% Optiprep, but is not limited thereto.

[0057] Next, the cell pellet is resuspended and plated.

[0058] Next, the plated cells are cultured in proliferation media.

[0059] According to one embodiment of the present invention, the proliferation medium may include, but is not limited to, DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) medium containing growth factors PDGF-AA, FGF, and EGF.

[0060] The method for culturing oligodendrocyte precursor cells according to the present invention can significantly increase the number of cells obtained by promoting the proliferation of oligodendrocyte precursor cells by optimizing the proliferation medium conditions.

[0061] According to one embodiment of the present invention, the growth medium may further include a supplement selected from the group consisting of, but not limited to, B27, GlutaMAX, Penicillin / Streptomycin, and combinations thereof.

[0062]

[0063] In addition, the second aspect of the present invention provides a method for culturing oligodendrocytes, comprising: a step of culturing oligodendrocyte precursor cells by the method according to the first aspect of the present invention; a step of isolating and replating the oligodendrocyte precursor cells by treating them with an enzyme; and a step of culturing the plated oligodendrocyte precursor cells in a proliferation medium.

[0064] Regarding the method for culturing oligodendrocytes according to the second aspect of the present invention, detailed descriptions of parts overlapping with the first aspect of the present invention have been omitted, but even if the descriptions have been omitted, the contents described in the first aspect of the present invention can be equally applied to the second aspect of the present invention.

[0065] In the oligodendrocyte culture method according to the present invention, by performing replating, it is possible to minimize irregular cell arrangement caused by the proliferation and migration ability of oligodendrocyte precursor cells and narrow intercellular gaps that cause cell death during differentiation.

[0066] According to one embodiment of the present invention, the enzyme may include, but is not limited to, one selected from the group consisting of Accutase, TrypLE, trypsin, and combinations thereof.

[0067] According to one embodiment of the present invention, the proliferation medium may include, but is not limited to, DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) medium containing growth factors PDGF-AA and FGF.

[0068] According to one embodiment of the present invention, the growth medium may further include a supplement selected from the group consisting of, but not limited to, B27, GlutaMAX, Penicillin / Streptomycin, and combinations thereof.

[0069]

[0070] In addition, the third aspect of the present invention provides a method for culturing myelin-forming oligodendrocytes, comprising the steps of culturing oligodendrocytes by the method according to the second aspect of the present invention; and the step of myelinating the oligodendrocytes by replacing the medium with a differentiation medium.

[0071] Regarding the method for culturing myelin-forming oligodendrocytes according to the third aspect of the present invention, detailed descriptions of parts overlapping with the first aspect and / or the second aspect of the present invention have been omitted. However, even if the descriptions have been omitted, the contents described in the first aspect and / or the second aspect of the present invention can be equally applied to the third aspect of the present invention.

[0072] According to one embodiment of the present invention, the differentiation medium may include, but is not limited to, a neurobasal medium containing growth factors N2 and T3.

[0073] The method for culturing myelin-forming oligodendrocytes according to the present invention optimizes the differentiation medium conditions, thereby obtaining more morphologically mature oligodendrocytes during the same differentiation period compared to the DMEM-based medium conditions that have been widely used in the past for differentiation into myelin-forming oligodendrocytes.

[0074] According to one embodiment of the present invention, the differentiation medium may further include a supplement selected from the group consisting of, but not limited to, B27, GlutaMAX, Penicillin / Streptomycin, and combinations thereof.

[0075] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0076]

[0077] [Example 1] Culturing of oligodendrocyte progenitor cells (OPCs)

[0078] The brains of P1 rats were collected in a 50-mL conical tube containing cold Hank's Balanced Salt Solution (HBSS), transferred to another tube containing cold 70% ethanol, and then transferred to a new tube containing cold HBSS. After dissecting the cerebral cortex and removing the meninges, the hemispheres were transferred to 15-mL conical tubes containing 200 μL of HBSS, one brain per tube (two hemispheres). 1 mL of AccumaxTM (Merck Millipore, #SCR006) was added to each tube, and the brain was triturated 10 times with a 1 mL pipette tip to prepare a suspension containing neural stem cells. 4 mL of isolation medium [Hibernate-A medium (Thermo Fisher Scientific, A1247501), 2% B-27 supplement (Thermo Fisher Scientific, #17504-044), 1% GlutaMAX (Thermo Fisher Scientific, #35050-061), 1% penicillin / streptomycin, 200 IU of papain (Worthington, #LS003126)] was added to each tube, and the tissue was dissociated in a shaking incubator at 30°C for 30 minutes.

[0079] Subsequently, the dissociated cell suspension was centrifuged at 200 g for 5 min at room temperature (RT), the supernatant was discarded, and the pellet was resuspended in 1 mL of isolation medium and 12% OptiprepTM (Sigma Aldrich, #D1556).

[0080] Next, the cell suspension was gently triturated 20 times using a 10 μL pipette tip attached to a 1 mL tip, 3 mL of separation medium and 12% Optiprep were added, and a total of 4 mL of cell suspension was centrifuged for the first time at 200 g for 15 minutes at room temperature.

[0081] Next, the supernatant was obtained, transferred to another 15 mL tube, diluted to 6% Optiprep by adding 4 mL of separation medium, and centrifuged a second time at 200 g for 15 minutes at room temperature.

[0082] Then, after removing the supernatant, the final pellet was resuspended in PBS and plated on a culture surface coated with poly-D-lysine (PDL, Sigma Aldrich, #P6407) 1*10 4 cells / cm 2 Plated with a density of .

[0083] Afterwards, oligodendrocyte progenitor cells were obtained by culturing them for 5-6 days in OPC proliferation medium under various conditions as shown in Table 1 below.

[0084] [Table 1] Proliferation medium conditions for culturing oligodendrocyte progenitor cells

[0085]

[0086] [Example 2] Culturing of oligodendrocytes (OL)

[0087] The medium was aspirated from the oligodendrocyte progenitor cells cultured in Example 1 (using the OPC proliferation medium of Condition 4 of Table 1 above), AccutaseTM (Thermo Fisher Scientific, #A11105-01) was added, and the flask was cultured for 5 minutes in a 37°C, 5% CO2 incubator, and then the cells were detached by gently tapping the flask.

[0088] Then, the cell suspension was transferred to a 15 ml tube and centrifuged at 200 g for 3 min at room temperature, and the resulting pellet was resuspended in PBS, counted, and 1Х10 4 cells / cm 2 were replated onto an appropriate culture surface at a density of .

[0089] Next, oligodendrocytes were obtained by culturing for 2 days in the proliferation medium of condition 3 of Table 1 above.

[0090] [Example 3] Culturing of myelin-forming oligodendrocytes

[0091] The medium of the oligodendrocytes cultured in Example 2 was replaced with a differentiation medium with various conditions as shown in Table 2 below, and the cells were differentiated for 2 or 4 days to obtain myelin-forming oligodendrocytes.

[0092] [Table 2] Differentiation medium conditions

[0093]

[0094]

[0095] [Example 4] Oligodendrocyte / neuron co-culture

[0096] Primary neurons were obtained from the cortex of P1 rats as follows.

[0097] First, a cell pellet was obtained through a primary centrifugation process using the separation medium [Hibernate-A medium (Thermo Fisher Scientific, A1247501), 2% B-27 supplement (Thermo Fisher Scientific, #17504-044), 1% GlutaMAX (Thermo Fisher Scientific, #35050-061), 1% penicillin / streptomycin, 200 IU of papain (Worthington, #LS003126)] and 12% Optiprep in the method of Example 1, and 5*10 4 dog cells / cm 2 were seeded on the culture surface at a density of .

[0098] Primary neuron cultures were generated by maintaining cells for 14 days and exchanging half of the medium every 3 days. On day 14, the medium was exchanged with differentiation medium of condition 5 in Table 2 above. Subcultured OPCs were seeded at 3*10 4 cells / cm 2Neurons were seeded at a density of 10 μM. Symbiotic cultures were maintained for 4 days, and only half of the medium was replaced on the second day.

[0099]

[0100] [Example 5] Culturing myelinated oligodendrocytes on aligned nanofibers

[0101] The passaged oligodendrocyte precursor cells were cultured at 3*10 in the growth medium of condition 3 of Table 1 above. 4 cells / cm 2 Cells were seeded onto 12 well-aligned nanofiber inserts (Sigma Aldrich, #Z694614-12EA, fiber diameter = 700 nm) at a density of 100 μm. After 2 days of proliferation, the medium was changed to differentiation medium of condition 5 in Table 2, and differentiation was maintained for 4 days with half the medium exchange on the second day after the change.

[0102]

[0103] [Example 6] WST-8 cell viability / proliferation assay

[0104] WST-8 assays were performed using OPCs cultured in 96-well plates using the Quantimax™ Cell Viability Kit (BioMax, #QM1000) according to the manufacturer's protocol. After isolation and proliferation for 72, 96, and 120 h, respectively, the medium was aspirated, the cells were washed with prewarmed PBS, and 100 μL of phenol red-free DMEM (Thermo Fisher Scientific, #21063029) was added. 10 μL of Quantimax was immediately added to each well, and the plates were incubated in a 5% CO2 incubator for 30 min. The optical density (OD) at 450 nm was measured using a microplate reader. Proliferation capacity was calculated as a relative value using the following formula: (450 nm OD of the well - average of the 450 nm OD values ​​of the blank) / (average of the 450 nm OD values ​​of Condition 1 in Table 1).

[0105]

[0106] [Example 7] Immunofluorescence

[0107] Cells cultured on 9 mm coverslips and nanofiber inserts were used for immunofluorescence. After the specified incubation period, cells were fixed with 4% paraformaldehyde for 20 min, washed three times with phosphate-buffered saline (PBS), and stored at 4°C until use. Cells were blocked with 10% normal goat serum (NGS) and 0.1% Triton X-100 in PBS for 1 h at RT. Cells were washed with PBS and incubated overnight at 4°C with the following primary antibodies in blocking solution:

[0108] anti-NG2 (1:500, Millipore, #AB5320), anti-A2B5 (1:500, Millipore, #MAB312), anti-MBP (1:500, Abcam, #AB7349), anti-Olig2 (1:500, Merck Millipore, #MABN50), anti-GFAP (1:2000, Dako, #Z033429), anti-GFAP (1:2000, Abcam, #AB4674), anti-Tuj1 (1:2000, Dako, #Z033429), anti-Iba1 (1:500, Wako, #019-19741), anti-cleaved-caspase-3 (1:200, Cell Signaling, #9664S), anti-Ki67 (1:500, Thermo Fisher Scientific, #MA514520).

[0109] Cells were washed with PBS and incubated with the appropriate Alexa Fluor 488-, 594-, or 680-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 hour at room temperature. Cells were then counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma Aldrich, #D9542) for 10 minutes at room temperature and mounted on glass slides. Cells were imaged using a Zeiss LSM800 confocal microscope.

[0110]

[0111] [Example 8] EdU proliferation assay

[0112] EdU proliferation assay was performed using the EdU Assay / EdU Staining Proliferation Kit (iFluor 488) (Abcam, ab219801) according to the manufacturer's protocol. Isolated OPCs were proliferated for 108 h and incubated with EdU for 2 h. Cells were fixed with 4% PFA and stained for Ki67 according to the immunofluorescence procedure. An additional EdU labeling step using an EdU reaction mixture containing iFluor 488 azide was applied between the secondary antibody incubation and DAPI counterstaining steps, with an incubation period of 30 min at room temperature.

[0113]

[0114] [Example 9] Statistics and Image Quantification

[0115] Independent t tests, one-way analysis of variance (ANOVA) with Tukey's multiple comparison test, simple linear regression, spline / LOWESS fitting, and frequency distribution analysis were performed using Graphpad Prism 9.5.1. A P value less than .05 was considered statistically significant. Cell counting was performed using Fiji / ImageJ with the particle analysis function, where applicable. Nearest neighbor distance (NND) measurements were obtained by first obtaining the XY coordinates of DAPI-stained cell nuclei images in Fiji / ImageJ, and then transforming the data table into a Poisson point process using the spatial statistics (spatstat) package in R (version 4.2.0). Nearest neighbor distances and spatial G-function results were obtained using spatstat. Sholl analysis was performed using the Neuroanatomy plugin in Fiji / ImageJ. Myelin processes of OLs visualized by immunostaining for MBP and binary masks were obtained by thresholding the images. A Sholl analysis was performed by marking the center point at the DAPI+ nucleus of the target cell, drawing circles with increasing radii centered on the center point, and quantifying the number of intersections between each circle and the binary mask. Morphological complexity was compared by comparing the total number of intersections in the cells. A schematic of the culture procedure was created using BioRender.com.

[0116]

[0117] [Experimental Example 1] OPC isolation

[0118] FIG. 1A is a schematic diagram of a culture method according to an experimental example of the present invention, FIG. 1B is a representative image of cells isolated after 5 days of proliferation (immunostaining for NG2 and A2B5, P: PDGF, F: FGF, E: EGF), FIG. 1C is a representative image of cells isolated after 5 days of proliferation (immunostaining for Tuj1, P: PDGF, F: FGF, E: EGF), and FIG. 1D is the result of quantification of the cell ratio after 5 days of proliferation according to the medium composition corresponding to the medium conditions of Table 1 [10 images per media condition (6 media conditions, n=3 samples), approximately 300 DAPI+ cells per image. One-way ANOVA and Tukey's multiple comparison test, ***: P < .001, ns: not statistically significant (P > .05)].

[0119] Density gradient centrifugation has a significant drawback: the amount of tissue that can be loaded onto a single column is substantially limited compared to other centrifugation methods, as cells and debris are concentrated in a single step during the process. To circumvent this problem, we utilized the variable sedimentation velocities of particles and adopted the principle of differential centrifugation, widely used for organelle fractionation or exosome isolation. Specifically, after dissecting and isolating the cerebral cortex of a P1 rat, the resulting cell suspension was subjected to two consecutive centrifugations using density gradient medium Optiprep to remove unwanted cells and debris (Fig. 1A).

[0120] First, the cell suspension was centrifuged in Hibernate medium-based isolation medium containing 12% Optiprep, and after centrifugation, the supernatant was transferred to another 15 ml tube, diluted with 6% Optiprep (volume 8 ml), and centrifuged again.

[0121] The cell population of the pellet obtained through secondary centrifugation was 1*10 4 cells / cm 2When plated at low density and cultured for 5 days in DMEM or DMEM / F12-based OPC proliferation medium, NG2 / A2B5+ OPC colonies comprised 90% to 95% of the total population and Tuj1+ neurons comprised 3% to 5% (Fig. 1 B, C, D).

[0122] GFAP+ astrocytes comprised approximately 0.5% of the total population, and Iba1+ microglia were not observed. In a neurobasal-based proliferation medium composition, OPC colonies were reduced in size and their proportion was low (approximately 75% to 77%).

[0123] Furthermore, Tuj1+ neurons comprised approximately 20% of the total population. The cell pellet from the first centrifugation contained erythrocytes, and when cultured using the same procedure, Iba1+ microglia were identifiable, but the cell populations were similar. Therefore, two sequential density centrifugations yielded a cell population enriched in OPCs after 5–6 days of expansion.

[0124]

[0125] [Experimental Example 2] Medium composition / growth factors for OPC proliferation

[0126] FIG. 2A is a representative image of cells isolated after 5 days of proliferation according to one experimental example of the present invention (staining for EdU, Ki67. P: PDGF, F: FGF, E: EGF), FIG. 2B is the quantification result of EdU+ / DAPI+ (%) cells according to the media composition corresponding to the media conditions of Table 1 [10 images per media condition (6 media conditions, n=3 samples), approximately 500 DAPI+ cells per image. One-way ANOVA and Tukey's multiple comparison test, ns: not statistically significant (P > .05)], and FIG. 2C is the quantification result of Ki67+ / DAPI+ (%) cells according to the media composition corresponding to the media conditions of Table 1 [10 images per media condition (6 media conditions, n=3 samples), approximately 500 DAPI+ cells per image. One-way ANOVA and Tukey's multiple comparison test, ns: not statistically significant (P > .05)], Figure 2D is a graph of the results of the WST-8 viability assay, values ​​for the DMEM (medium composition 1) group cultured for 72 hours (6 medium conditions, n=3 samples, one-way ANOVA and Tukey's multiple comparison test, ***: P < .001).

[0127] After isolating OPCs, we compared the compositions of various OPC proliferation media to determine the most optimal formulation for inducing OPC proliferation. Supplement B27, which contains various nutrient factors and is frequently used in serum-free oligodendrocyte culture media, was used in all conditions, as were growth factors PDGF and FGF, known to induce OPC proliferation and inhibit differentiation. Six conditions were compared, as shown in Table 1, based on the presence or absence of DMEM, DMEM / F12, and Neurobasal media, which are commonly used as basic media for primary OL culture, and EGF, which has been shown to promote OPC proliferation in vitro.

[0128] Isolated OPCs proliferated for 72–120 hours under the specified conditions. Immunostaining for proliferation markers EdU and Ki67 showed no differences in cell proliferation rates between media conditions, but WST-8 proliferation / viability assays revealed that condition 4 (DMEM / F12 with EGF) performed best at both 96 and 120 hours, demonstrating approximately 10–15 times higher viable cell counts compared to the neurobasal-based formulation (Fig. 2B, C, and D).

[0129] Therefore, DMEM / F12 containing EGF, condition 4 in Table 1, was selected as the optimal OPC proliferation medium, and the following steps were performed using OPCs proliferated in this medium composition throughout the culture process.

[0130]

[0131] [Experimental Example 3] Differentiation of OPCs into OLs and passage of the proliferated OPCs

[0132] FIG. 3A is a representative image of cells isolated 4 days after differentiation according to one experimental example of the present invention (immunostaining for myelin basic protein (MBP)), FIG. 3B is a representative image of cells isolated at the periphery of colonies 4 days after differentiation (region B of FIG. 3A; immunostaining for cleaved caspase 3, MBP), FIG. 3C is a representative image of cells isolated at the center of colonies 4 days after differentiation (region C of FIG. 3A; immunostaining for cleaved caspase 3, MBP), and FIG. 3D is a scatter plot of the average nearest neighbor distance of cells in the immunostaining images and the percentage of cleaved caspase-3+ / DAPI+ cells (%) and the percentage of cell death measured by spline / LOWESS fitting. The correlation between average nearest neighbor distance and cell death rate was analyzed by simple linear regression for each media condition [10 images per media condition (6 media conditions, n=3 samples), approximately 300 DAPI+ cells per image].

[0133] After successful proliferation of isolated OPCs, the culture medium was replaced with OL differentiation medium. This time, various medium compositions were tested to differentiate OPCs into mature OLs. The use of B27 and N2 supplements, which contain insulin-like growth factor (IGF), known to be essential for the survival of mature OLs in vitro, and thyroid hormone T3, a well-known OL differentiation promoter, was consistent across all conditions.

[0134] In a similar manner to the OPC proliferation medium conditions, we tested another six conditions that could be distinguished by the basal medium as shown in Table 2 above. After 4 days of differentiation, MBP+ mature oligodendrocytes with web-like myelin sheets could be observed in all groups (Fig. 3B). However, in addition to the successful differentiation of OPCs into mature OLs, we also found clumped oligodendrocyte areas with indistinguishable tangled MBP+ processes, which showed nuclear condensation and immunoreactivity for cleaved caspase 3, indicating that they had undergone apoptosis (Fig. 3C).

[0135] When visualized at low magnification, these apoptotic OL clumps were most prominent in the center of the colonies, where focal cell density was highest (Fig. 3A). To confirm the association between high cell density and the average nearest neighbor distance of cells in images containing 100–600 cells per image, we analyzed the correlation between the percentage of cleaved caspase-3-positive apoptotic cells and the average nearest neighbor distance. We found an inverse correlation between the average nearest neighbor distance and cell death rate, suggesting that overcrowding of OPCs during proliferation leads to cell death during maturation induction (Fig. 3D). We concluded that additional procedures were necessary to redistribute the diffused OPCs and secure more space.

[0136] FIG. 4A is a schematic diagram of a revised culture procedure including passage of expanded OPCs according to one experimental example of the present invention, FIG. 4B is a representative image of passaged and redistributed OPCs (immunostaining for Olig2; arrows indicate Olig2+ cells with atypical morphology), FIG. 4C is a quantification of Olig2+ / DAPI+ (%) cells according to media compositions corresponding to the media conditions in Table 1 [(10 images per media condition (6 media conditions, n=3 samples), approximately 50 DAPI+ cells per image, one-way ANOVA and Tukey's multiple comparison test, ns: not statistically significant (P > .05)), FIG. 4D is a representative histogram of nearest neighbor distance (NND) of OPCs after passage [10 images per media condition (6 media conditions, n=3 samples), approximately 200 DAPI+ cells per image], and FIG. 4E is a representative histogram of proliferation media. Here is a plot of the spatial G-function (cumulative distribution of nearest neighbor distances) of passaged OPCs by composition (gray area indicates the boundary of complete spatial randomness), Fig. 4F is a representative low-magnification image of passaged OPCs after 4 days of differentiation into mature oligodendrocytes in neurobasal-based differentiation medium (condition 5 in Table 2; immunostaining for cleaved caspase 3, MBP), and Fig. 4G is a scatterplot of the mean nearest neighbor distance of cells in the immunostained images versus the percentage of cleaved caspase 3+ / DAPI+ cells and the percentage of apoptosis measured by spline / LOWESS fitting. The correlation between mean nearest neighbor distance and percentage of apoptosis was analyzed by simple linear regression for each media condition [9 images per media condition (6 media conditions, n=3 samples), approximately 200 DAPI+ cells per image].

[0137] To redistribute and space OPCs to avoid cell death during differentiation, proliferated OPCs were passaged and redistributed at 1*10 4 cells / cm 2were seeded at a density of 10 μM (Fig. 4A). The passaged OPCs were stabilized in OPC growth medium for 2 days. The previous six growth medium conditions were retested to determine which condition best suited the 2-day stabilization period.

[0138] During OPC proliferation after passage, we observed that Tuj1+ neurons, which previously comprised 5% of the total population, now decreased to almost non-existent, and that the purity of Olig2-positive OL lineage cells was determined to be OPCs with bipolar / tripolar morphology.

[0139] Immunoreactivity for NG2 increased from 90% to 95% to approximately 98% (Figures 4B, 4C). The average nearest neighbor distance of passaged cells increased from approximately 15 μm to 19 μm, and the average apoptosis rate, measured as the percentage of cleaved caspase-3-positive cells, decreased from 13% to 4% (Figures 4F, 4G). When DMEM / F12 with EGF, the selected condition during OPC proliferation, was used, we observed that cells had a more heterogeneous spatial distribution compared to other media conditions. To quantify the "randomness" of the spatial distribution, the coordinates of cell nuclei, represented by DAPI in square images, were transformed using a Poisson point process and a histogram of the nearest neighbor distance and a G-function, the cumulative distribution function of the nearest neighbor distance, were generated. Whereas cells stabilized after passage in the other five media conditions were distributed within the boundaries of complete spatial randomness, cells stabilized in DMEM / F12 with EGF were shifted to the left in both the histogram of the NND frequency distribution and the G function, indicating clustering of cells (Figures 4D, 4E). Cells exhibiting morphology different from typical OPCs were also observed in media conditions containing EGF (especially when combined with neurobasal media), and therefore DMEM / F12 without EGF (condition 3 in Table 1) was chosen as the medium for the post-passage stabilization period (Figure 4B).

[0140]

[0141] [Experimental Example 4] Differentiation of passaged OPCs

[0142] FIG. 5A is a representative image of differentiated oligodendrocytes after passage for 2 or 4 days in the designated differentiation medium composition according to one experimental example of the present invention (immunostaining for MBP), FIG. 5B is a bar graph of the cell composition of differentiated oligodendrocytes according to the medium composition corresponding to the medium conditions of Table 2, and FIG. 5C is the result of Sholl analysis of differentiated oligodendrocytes [Comparison of morphological complexity was performed by comparing the total number of intersections per cell; 10 images per media condition (6 media conditions, n=3 samples); approximately 100 MBP+ OL per image; one-way ANOVA and Tukey's multiple comparison test; ****: P < .0001; ns: not statistically significant (P > .05)].

[0143] After the passaged OPCs stabilized and proliferated for 2 days, the six differentiation medium conditions listed in Table 2 were tested again. After 2 days of differentiation, MBP-positive mature OLs with branched processes, which comprised approximately 40–50% of the population, were observed regardless of the medium used (Figures 5A, 5B). By day 4, spider-like myelin sheets were observed in all groups. Despite the differences in maturation levels, the actual number of MBP+ OLs did not differ significantly between cultures differentiated for 2 and 4 days (Figures 5A, 5B). 15–20% of the cells consistently remained NG2+ OPCs across all medium conditions (Figure 5A). Sholl analysis was performed to compare the degree of differentiation according to morphological complexity. The results showed that the neurobasal medium-based condition generated OLs with significantly more complex processes by day 4 (Figure 5C). The use of CNTF did not affect OL maturation in terms of number or morphological complexity (Figs. 5A, 5B).

[0144] Meanwhile, we observed that OPCs converted into GFAP-positive astrocytes during differentiation, accounting for approximately 15% of the cell population on day 2 and not increasing in number until day 4. The media conditions did not affect the proportion of astrocytes, but GFAP-positive astrocyte processes appeared more enlarged under conditions where CNTF was used (Fig. 5B). Through this, we successfully induced differentiation of OPCs into mature OLs and found that the Neurobasal-based differentiation medium significantly enhanced the morphological complexity of mature OLs.

[0145]

[0146] [Experimental Example 5] Myelination of cultured OPCs

[0147] Figure 6A is a representative image of a neuron / oligodendrocyte co-culture according to an experimental example of the present invention (immunostaining for MBP, Tuj1), and Figure 6B is a representative image of differentiated oligodendrocytes cultured on aligned nanofibers (immunostaining for MBP).

[0148] After confirming successful OL differentiation, the ability of OLs obtained by the culture method according to the present invention to envelop neurons or neuron-like structures and form myelin sheaths was verified.

[0149] After observing the presence of neurons during OPC proliferation after isolation, 5*10 were seeded in Neurobasal medium supplemented with B27 and N2 without OPC preferring growth factors PDGF and FGF. 4 cells / cm 2 Primary neuron cultures were obtained by seeding cells isolated from the second centrifugation step using 6% Optiprep at a high density of 3*10 4 cells / cm 2OPCs passaged in neuronal cultures at a density of 100 μM were seeded, and the differentiation medium was changed to Condition 5 (Neurobasal-based medium without CNTF) in Table 2. On day 4 of differentiation, we found that areas of overlapping Tuj1-positive axons and MBP-positive myelin sheets indeed existed (Fig. 6A).

[0150] Furthermore, the ability of cultured OLs to encapsulate aligned nanofibers was also investigated. The entire culture process from isolation to differentiation was repeated, except that passaged OPCs were seeded onto 12-well aligned nanofiber inserts with a diameter of 700 nm. Z-stack images of MBP-positive myelin sheaths on aligned fibers were captured, visualized with a differential interference contrast (DIC) filter, and efficient myelin sheath formation was confirmed in 3D reconstructions surrounding aligned fibers (Fig. 6B).

[0151] Therefore, it was confirmed that OLs cultured through the culture method according to the present invention can form myelin sheets in vitro and surround neurons.

[0152]

[0153] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0154] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A step of dissociating tissue by adding isolation media to a suspension containing neural stem cells; A step of resuspending the dissociated cell suspension by adding the first solution; A step of adding the first solution to the resuspended solution and performing the first centrifugation to obtain a supernatant; A step of adding a second solution to the above supernatant and performing a second centrifugation to obtain a cell pellet; A step of plating after resuspending the above cell pellet; and A step of culturing plated cells in proliferation media; including, Method for culturing oligodendrocyte precursor cells.

2. In paragraph 1, The above separation medium comprises Hibernate-A medium, GlutaMAX, Penicillin / Streptomycin and papain. Method for culturing oligodendrocyte precursor cells.

3. In paragraph 1, The first solution comprises a separation medium and high-density iodixanol, Method for culturing oligodendrocyte precursor cells.

4. In paragraph 1, The second solution comprises a separation medium and low-density iodixanol, Method for culturing oligodendrocyte precursor cells.

5. In paragraph 1, The above proliferation medium is DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) medium containing growth factors PDGF-AA, FGF and EGF. Method for culturing oligodendrocyte precursor cells.

6. In paragraph 5, The above growth medium further comprises a supplement selected from the group consisting of B27, GlutaMAX, Penicillin / Streptomycin and combinations thereof. Method for culturing oligodendrocyte precursor cells.

7. A step of culturing oligodendrocyte precursor cells by the method according to paragraph 1; A step of isolating and replating the above oligodendrocyte precursor cells by treating them with an enzyme; and A step of culturing plated oligodendrocyte precursor cells in a proliferation medium; including, Method for culturing oligodendrocytes.

8. In paragraph 7, The enzyme comprises one selected from the group consisting of Accutase, TrypLE, trypsin and combinations thereof. Method for culturing oligodendrocytes.

9. In paragraph 7, The above proliferation medium is DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) medium containing growth factors PDGF-AA and FGF. Method for culturing oligodendrocytes.

10. In paragraph 9, The above growth medium further comprises a supplement selected from the group consisting of B27, GlutaMAX, Penicillin / Streptomycin and combinations thereof. Method for culturing oligodendrocytes.

11. A step of culturing oligodendrocytes by the method according to Article 7; and A step of myelinizing the oligodendrocytes by replacing the medium with a differentiation medium; including, Method for culturing myelin-forming oligodendrocytes.

12. In paragraph 11, The above differentiation medium contains growth factors N2 and T3 in neurobasal medium. Method for culturing myelin-forming oligodendrocytes.

13. In paragraph 12, The above differentiation medium further comprises a supplement selected from the group consisting of B27, GlutaMAX, Penicillin / Streptomycin, and combinations thereof. Method for culturing myelin-forming oligodendrocytes.

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